Common antigen (PSC-A) to Pseudomonas aeruginosa which acts as an agent for pretecting Pseudomonas aeruginosa infection

ABSTRACT

PSC-A is a new serological common antigen to Pseudomonas aeruginosa having very low toxicity, and highly effective for protecting infection by any of the serotypes of Pseudomonas aeruginosa. PSC-A can be used as the active component in a pharmaceutical agent for protecting Pseudonomas aeruginosa infection.

FIELD OF THE INVENTION

This invention relates to PSC-A, a new common antigen to Pseudomonasaeruginosa (hereafter abbreviated as P. aeruginosa) and is effective forprotecting P. aeruginosa infection.

BACKGROUND OF THE INVENTION

P. aeruginosa is originally known as an attenuated pathogenic bacterium.In accordance with the recent increase in the rate of occurrence ofbacterial replacement and proliferation resulting from theadministration of large doses of antibiotics, the number of patientsinfected by P. aeruginosa as distinct from other bacterical infectiousdiseases has notably increased.

P. aeruginosa easily causes infectious disease in patients havingreduced resistance to infection, and therefore this infectious diseaseis one of the representative opportunistic infectious diseases. Theabove patients having reduced resistance to infection include patientswith cancer, those receiving immunosuppressive therapy, implantationpatients, burn patients, and infants.

After once occuring, the acute disease caused by this bacterum has astrong tendency to develop into a systemic infection. Also a goodprognosis for this disease can not be expected. Therefore this diseasehas come to be numbered as one of the most difficient to cure curedbacterial infectious diseases.

Infectious diseases caused by P. aeruginosa are generally difficult tocure for the following reasons:

(1) This bacterium exhibits a high resistance to almost all of theantibiotics which have been typically used.

(2) This bacterium tends to be resistant to recently developedantibiotics which are effective against P. aeruginosa.

(3) There is a high incidence of infectious diseases caused by P.aeruginosa which can not be satisfactorily treated by chemotherapy usingthese antibiotics.

On the basis of the fact that there is a limit to the chemotherapy ofinfectious diseases caused by P. aeruginosa performed using antibiotics,several attempts have been made to develop vaccines orinfection-protective agents which are prepared from cell components ofP. aeruginosa and used to increase the ability of the host to protectitself against P. aeruginosa.

For instance, the polyvalent vaccine PEV-01 protective against allserotypes of P. aeruginosa was prepared by purifying the superficialantigen of each serotype of P. aeruginosa (specific antigen for eachserotype) and mixing the purified superficial antigens (Lancet II; 977,1979). After performing an attempt to clinically apply this polyvalentvaccine to burn patients, it was reported that this vaccine isprotective against septicemia by P. aeruginosa infection.

However, the preparation of this polyvalent vaccine is complicated bymixing more than 10 antigens against different serotypes of P.aeruginosa. Besides, since the specific antigens of different serotypeswhich are components of this vaccine are so-called O antigens existingin the surface layers of cells of P. aeruginosa and are composed oflipopolysaccharides called endotoxin, this vaccine cause unavoidablylocal and systemic side effects such as intense pyrogenecity.

OEP (original endotoxin protein) principally composes of protein wasisolated as a serological common antigen to P. aeruginosa by Homma etal. It was reported that OEP works as an protective antigen common toall serotypes of P. aeruginosa and can be used for vaccine (Jpn. J. Exp.Med.; 47, 393-402, 1977).

Homma et al. thought that this infection can not satisfactorily beprotected by OEP alone and carried out each clinical experiment by avaccine consisting of a mixture of toxoids prepared by combining OEPwith two or three components among protease, elastase and exo toxoidfrom P. aeruginosa. Although it was reported that these vaccines showedgood effect in some cases, they have not yet been put to practical use.

The other vaccines of P. aeruginosa include ribosome vaccines eachhaving a specific effect against each serotype of the bacterium, pilivaccines characterized by adhering to the surface of membrane, andflagella vaccines which stop the movement of bacteria. However, all ofthese vaccines are still in the experimental stage. Therefore, it is noexaggeration to say that the appearance of excellent vaccines against P.aeruginosa infection is still desired.

SUMMARY OF THE INVENTION

During investigations which have been performed to find a newinfection-inhibiting antigen which by itself has the ability to protectagainst P. aeruginosa infection and acts upon all serotypes of P.aeruginosa, the inventors succeeded in preparing a mouse-hybridoma whichproduces a monoclonal antibody which can react with serotypes of P.aeruginosa. Additionally, the authors first discovered that the cellcomponent of P. aeruginosa corresponding to the above monoclonalantibody has great ability to protect infection by all serotype of P.aeruginosa.

Specially, during a study performed to prepare a hybridoma which canproduce an anti-P. aeruginosa monoclonal antibody through the cellfusion between spleen cells of mice immunized with P. aeruginosa andmouse myeloma cells, the inventors succeeded in producing a hybrid cellline which can produce a monoclonal antibody to react with all serotypesof P. aeruginosa (hereafter referred to as C-Ab-producing hybridoma).After that, the inventors isolated and purified the cell component of P.aeruginosa corresponding to the monoclonal antibody produced by theC-Ab-producing hybridoma (hereafter referred to as C-Ab) by an affinitychromatography using immobilizing C-Ab. The common antigen obtained thusto P. aeruginosa which can react with C-Ab was found to be a completelynew substance composed of protein, has ver low toxicity, and exhibitsexcellent ability in an animal experiment to protect infection by allserotypes of P. aeruginosa, resulting in this invention.

The substance of this invention will be simply referred to as PSC-Ahereafter.

This invention relates to PSC-A, a new common antigen to P. aeruginosawhich is protective against P. aeruginosa infection. More specifically,this invention relates to the new common antigen, PSC-A, which isobtained from P. aeruginosa, and has the ability to protect P.aeruginosa infection as well as the preparation of PSC-A and an agentfor protection against P. aeruginosa infection.

This invention also relates to a method for preparing the common antigenPSC-A to P. aeruginosa having the ability to protect P. aeruginosainfection from a cell-free extract, which is obtained by crushing P.aeruginosa before centrifuging, through isolation and purification usingan affinity chromatography by immobilizing the monoclonal antibody witha specific affinity for the common antigen PSC-A.

This invention also relates to an agent for protecting P. aeruginosainfection which contains the common antigen PSC-A as its activecomponent.

The purpose of this invention is to supply an agent for protecting P.aeruginosa infection, which contains the common antigen PSC-A from P.aeruginosa as its active component for the vaccinotherapy of animals andhumans.

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE shows an ultraviolet spectrum for PSC-A.

DETAILED DESCRIPTION OF THE INVENTION

This invention will be concretely described in the following.

All strains of P. aeruginosa used in this invention are listed accordingto serological classification in Table 1. There are different theoriesabout the classification of P. aeruginosa. The strains of P. aeruginosaused in this invention, are classified according to serologicalclassification given in the report of the serotyping committee of theJapan P. aeruginosa society (1975, Japan J, Exp. Med., 46, 329, 1976).All strains belonging to A˜M groups according to this classification canbe used as a specific serotype of P. aeruginosa described in thisinvention. Since it was thought to be the best method, theclassification of P. aeruginosa for this invention was performedaccording to the serological classifications established by theserotyping committee. Considering that new classification criteria willbe adopted in the future, it can be said that the strains of P.aeruginosa which can be used in this invention, include all bacterialstrains having PSC-A as an infection-protecting antigen.

Thus, the bacterial strains which can be used in this invention are notrestricted to those belonging to the above groups A˜M or those which canbe classified according to this classification criteria. Here, among thestrains of P. aeruginosa belonging to groups A˜M and listed in table 1,most of the IID strains are registered in the American Type cultureCollection (catalogue of BACTERIA-PHAGES-γDNA VECTORS 16th edition, page136, 1985), that is an American organization for preserving bacterialstrains, and can be supplied from this organization.

                  TABLE 1                                                         ______________________________________                                        Classification established                                                    by Japan P. aeruginosa                                                        society       Strain                                                          ______________________________________                                        A P. aeruginosa                                                                             IID 1001 (ATCC 27577)                                           B P. aeruginosa                                                                             IID 1002, ATCC 27578, IID 1007                                                ATCC 27583, IID 1013, ATCC 27589,                                             IID 5004                                                        C P. aeruginosa                                                                             IID 1003, ATCC 27579, IID 1037,                                               IID 1021                                                        D P. aeruginosa                                                                             IID 1004 (ATCC 27580)                                           E P. aeruginosa                                                                             ATCC 27581, IID 1130, PA 103                                    F P. aeruginosa                                                                             IID 1006 (ATCC 27582)                                           G P. aeruginosa                                                                             ATCC 10145, ATCC 27584, P 28                                    H P. aeruginosa                                                                             IID 1009 (ATCC 27585)                                           I P. aeruginosa                                                                             ATCC 27586                                                      J P. aeruginosa                                                                             IID 1011 (ATCC 27587)                                           K P. aeruginosa                                                                             IID 1012 (ATCC 27588)                                           L P. aeruginosa                                                                             IID 1014 (ATCC 27590)                                           N P. aeruginosa                                                                             IID 5018                                                        ______________________________________                                    

The preparation and the property of the monoclonal antibody C-Ab havinga specific affinity for PSC-A and used in an affinity chromatographyused to isolate and purify PSC-A, will be described in ExperimentalExample 1.

EXPERIMENTAL EXAMPLE 1 PRODUCTION, PREPARATION AND PROPERTY OFMONOCLONAL ANTIBODY C-Ab

C-Ab-producing hybridoma was produced according to the well-known methodby Kohler, Milstein et al. (Nature, 256, 495, 1975). After an emulsionof P. aeruginosa ATCC 27581 (E type) treated with 0.3% of formalin wasprepared with Freund incomplete adjuvant, this emulsion wasintraperitoneally administered to female BALB/C mouse (7 weeks old)every other week for a total of 5 times to achieve immunization. The5×10³ mouse spleen cells collected 4 days following the finalimmunization and 5×10⁷ NS-1 mouse myeloma cells were subjected to cellfusion in the presence of 50% polyethylene glycol to produce ahybridoma. The hybridoma thus prepared was then poured into a 96-wellflat bottom microplate and cultured on Dulbecco MEM medium supplementedwith 10% fetal bovine serum containing HAT (hypoxanthine, amimopterinand thymidine) at 37° C. in the presence of 5% of CO₂. For wells inwhich proliferation of the hybridoma was observed, the presence ofanit-P. aeruginosa monoclonal antibody in the culture solution wasdetermined by Dot Immunobinding Assay which is an enzyme immunoassay(Anal. Biochem. 119, 142-147, 1982, hereafter referred to as DIBAmethod). DIBA method was performed using a 96-well microtiter plateaccording to the following procedure. A nitrocellulose membrane filter(3.1 mm square) prepared by immobilizing 0.4 μg per dot of P. aeruginosatreated with 0.3% of formalin (used as the antigen) was incubated with100 μl of the above culture solution at room temperature for 30 minutesbefore being incubated with peroxidase-labelled anti-rabbit mouseimmunoglobulin antibody (manufactured by DAKO company) for 30 minutes.The nitrocellulose membrane filter was then reacted with4-chloro-1-naphtol as a substrate for peroxidase. A positive result wasrecorded when dark blue spot was observed on the membrane filter byimmobilizing the antigen. When the production of the monoclonal antibodywas detected in the culture solution, the hybridoma was furthersubjected to cloning by limiting dilution. The monoclonal hybridomaobtained thus was proliferation in a flask, and the proliferatedmonoclonal hybridoma was implanted in the abdominal cavity of a DALB/Cmouse treated with the immunosuppressant Pristane (Aldrichi company).Thus ascites fluid of the treated mouse was applied to an affinitychromatography with Protein A-Sepharose (Pharmacia company) to prepare apurified monoclonal antibody. Among the hybridomas obtained thus whichcan produce various anti-P. aeruginosa monoclonal antibodies, theinventors found a C-Ab-producing hybridoma which can produce amonoclonal antibody C-Ab reacting with all the serotypes of P.aeruginosa. The reactivities of this C-Ab for several serotypes of P.aeruginosa determined by DIBA method are shown in Table 2. This C-Abexhibited almost equal affinities for all serotypes of P. aeruginosa.Additionally, this C-Ab did not react with either the endotoxin fromserotype E of P. aeruginosa [lipopolysaccharied, prepared from the ATCC27581 strain (N-10 strain) of P. aeruginosa by the Morrison method (J.Biol. Chem., 250, 2911, 1977)], or OEP [prepared from the ATCC 27581strain (N-10 strain) of P. aeruginosa by the method of Homma et al.(Japan J. Exp. Med., 42, 23, 1972)]. This result indicates that PSC-Ahas a property different from those of these substances.

The monoclonal antibody C-Ab shown in the above Experimental Example 1,as will be described later in detail, is essential for purification toachieve simple high-yield of PSC-A from P. aeruginosa. It is usual thatC-Ab can be preferablly used after being immobilized with a carrier suchas Affigel-10 (Bio-Rad company) or BrCN-Sepharose (Pharmacia company).

The antibody for the preparation of PSC-A is not restricted only to C-Abshown in Experimental Example 1, and any antibodies having a specificaffinity for PSC-A can be favorably used for the method of thisinvention. For example, monoclonal antibodies can be used for affinitychromatography, each having a specific affinity for PSC-A and producedby either a hybridoma through the cell fusion between myeloma cells andanti-PSC-A-antibody-producing B lymphoid cells from an animal newlyimmunized with PSC-A isolated in Examples 1˜3 or an animal or humanstimulated with PSC-A in vitro, or proliferation-type cells by infectinghuman B lymphoid cells to produce anti-PSC-A antibody with EB virus.Besides, mixtures of these antibodies as well as anti-PSC-A polyclonalantibody obtained from the serum of an animal immunized with PSC-A canalso be used as antibodies for this invention.

                  TABLE 2                                                         ______________________________________                                                                  Titer of C-Ab                                                                 determined                                          Strain           Serotype by DIBA method                                      ______________________________________                                        P. aeruginosa IID1001                                                                          A        5.sup.5 × 10.sup.2                            P. aeruginosa IID1002                                                                          B        5.sup.4 × 10.sup.2                            P. aeruginosa IID1007                                                                          B        5.sup.4 × 10.sup.2                            P. aeruginosa IID1013                                                                          B        5.sup.4 × 10.sup.2                            P. aeruginosa IID5004                                                                          B        5.sup.4 × 10.sup.2                            P. aeruginosa IID1037                                                                          C        5.sup.4 × 10.sup.2                            P. aeruginosa IID1004                                                                          D        5.sup.4 × 10.sup.2                            P. aeruginosa IID1130                                                                          E        5.sup.4 × 10.sup.2                            P. aeruginosa ATCC27581                                                                        E        5.sup.4 × 10.sup.2                            P. aeruginosa PA103                                                                            E        5.sup.4 × 10.sup.2                            P. aeruginosa IID1006                                                                          F        5.sup.4 × 10.sup.2                            P. aeruginosa ATCC27584                                                                        G        5.sup.4 × 10.sup.2                            P. aeruginosa ATCC10145                                                                        G        5.sup.4 × 10.sup.2                            P. aeruginosa P-28                                                                             G        5.sup.4  × 10.sup.2                           P. aeruginosa IID1009                                                                          H        5.sup.4 × 10.sup.2                            P. aeruginosa ATCC27586                                                                        I        5.sup.4 × 10.sup.2                            P. aeruginosa IID1011                                                                          J        5.sup.4 × 10.sup.2                            P. aeruginosa IID1012                                                                          K        5.sup.4 × 10.sup.2                            P. aeruginosa IID1014                                                                          L        5.sup.4 × 10.sup.2                            P. aeruginosa IID5018                                                                          M        5.sup.4 × 10.sup.2                            ______________________________________                                         *1 Microbial cell from each serotype of P. aeruginosa treated with 0.3% o     formalin was dotted on a nitrocellulose membrane filter.                      *2 Classification of Japan P. aeruginosa Society.                             *3 CAb-producing hybridoma was implanted in the abdominal cavity of a         BALB/C mouse (8 week old) to obtain ascites fluid, and then the ascites       fluid was subjected to centrifugation (30000 × g, 20 min, 4°     C.) and supernatant was used for the activity.                                The antibody titer of CAb for each strain was expressed as the maximum        multiple of dilution on CAb (supernatant of ascites) for which dark blue      spot was observed on a nitrocellulose membrane filter.                   

Next, a basic method for producing PSC-A will be described. Strains ofP. aeruginosa which can be used in producing PSC-A are as describedabove. Conventional methods for culturing P. aeruginosa and crushingmicrobial cells may be used. For the medium, heart infusion broth, brainheart infusion broth (manufactured by Eiken Kagaku), nutrient broth, ora synthetic medium prepared according to the method of Homma et al. (J.Biochem. 83, 711-18, 1978) can be used. The synthetic medium by Homma etal. without any proteins is specially preferable because there is nopossibility for contamination of proteins in the medium into microbialcomponent. It is preferable that the temperature of the medium remainbetween 25° C. and 37° C., and that its pH be between 6.5 and 8.5. Theculture should be performed under aerobic conditions. For example, it isrecommended that shaking culture or aerational agitation culture in aculture vessel should be performed. The culture period influences theyield of PSC-A from P. aeruginosa.

Usually a culture period of 16˜24 hours is preferable. For example,after shaking culture at 37° C. for 24 hours on the synthetic medium(pH7.4) of Homma et al. microbial cells desired were obtained bycentrifugation of filtration. Here, it is possible to recover thesemicrobial cells after they are deactivated by being treated with a smallamount of formalin. The microbial cells obtained thus are thensufficiently mixed with water or a proper buffer solution before beingcrushed by a DYNOMILL, a French press, an ultrasonicator while beingcoolled below 10° C. The suspension obtained thus is then decanted andsubjected to centrifugation to obtain a cell-free extract of PSC-A. Thecentrifugation of the suspension is performed at 39,000×g for 30minutes, and the supernatant is collected. In preparing a suspension bycrushing microbial cells of P. aeruginosa by a machine, the rate for theextraction of PSC-A can be further improved by adding either a smallquantity of a surfactant such as Triton X-100 a chelating agent such asEDTA or an enzyme such as lysozyme, deoxyribonuclease or ribonuclease.

The cell-free extract used in this invention refers to a fraction whichis prepared by removing noncrushed microbial cells, partially crushedmicrobial cells, and the insoluble fraction of cell wall fraction fromthe suspension by centrifugation as much as possible. Crude PSC-A can beobtained from the cell-free extract by subjecting it to an ion-exchangecolumn chromatography or a gel filtration chromatography. PSC-A can beeasily obtained with a high yield according to the method used in thisinvention by isolating and purifying PSC-A by applying the cell-freeextract to an affinity column prepared by immobilizing a monoclonalantibody such as C-Ab. It is preferable that a monoclonal antibody suchas C-Ab with a specific affinity for PSC-A is used for the isolation andpurification of PSC-A is used after immobilized with a proper carriersuch as Affigel or CNBr-Sepharose as previously mentioned. The affinitycolumn to which the cell-free extract was applied is then thoroughlywashed with a neutral buffer with a pH of 6˜8 to elute contaminantsother than PSC-A which are not bound to the above immobilized monoclonalantibody. Next, a buffer of low pH range usually used for thedissociation of the antigen-antibody complex such as 50 mM glycine-HClbuffered physiological saline (pH 3.0) is used for the affinity columnto dissociate and elute PSC-A bound to the monoclonal antibody. Theeluate prepared thus is then adjusted pH to be neutral before theneutralized solution is dialyzed against distilled water. After that,the dialysate is lyophilized to obtain a pure powder of PSC-A.

The concentration of PSC-A in the filtrate from the culture can beneglected as long as microbial cells are obtained as a usual cultureproduct of living bacteria. However, when a large amount of PSC-A hasbeen transferred from microbial cells to a filtrate due to death orautolysis of microbial cells by a long period culture, it is possible torecover the purified PSC-A by applying a supernatant by centrifugationof the above filtrate to an affinity column prepared by immobilizing theabove monoclonal antibody with a specific affinity for PSC-A.

In order to effectively elute and remove contaminants other than PSC-Afrom the affinity column to which the previous mentioned cell-freeextract has been applied, washing the column with a neutral buffer witha small quantity of a surfactant such as Triton X100 is recommended.

The biophysical chemical properties of PSC-A obtained throughpurification in Example 1 will be shown in the following.

PSC-A obtained in Example 2 or Example 3 also exhibited the sameproperties.

Biophysical Chemical Properties of PSC-A Which is a Common Antigen to P.aeruginosa

(1) Molecular Weight

About 15,000 (by SDS-polyacrylamide gel electrophoresis) A single bandstained by coomassie brilliant blue R250 was observed in polyacrylamideelectrophoresis (PAGE) performed under each of the following conditions.

    ______________________________________                                        Conditions for PAGE                                                                            Result                                                       ______________________________________                                        pH 9.5         7% gel  Rf = 0.86                                              pH 9.5        10% gel  Rf = 0.80                                              pH 9.5        12% gel  Rf = 0.78                                              ______________________________________                                    

(2) Protein, Sugar and Hexosamine Content

Protein Content (%) : 55.0 [(colorimetric analysis by hydrolysis usingninhydrin, bovine serum albumin as the standard)(Anal. Biochem., 49, 95,1972)] 27.0 [modified Lowry method, bovine serum albumin as thestandard) (Anal. Biochem., 69, 646, 1975)], 24.0 [(protein bindingassay, bovine serum albumin as the standard)(Anal. Biochem., 72, 248,1976)].

Sugar Content (%) : 3.0˜5.0 [(phenol-sulfuric acid method, glucose asthe standard (Anal. Chem., 28, 350, 1956)].

Hexosamine Content (%) : below 1.0 [(Rondle. Morgan method, glucosamineas the standard)(Biochem. J., 61, 586, 1955)].

(3) Lipid Content (%) : 7.0˜10.0 [(modified Bligh. Dyer method (Can. J.Biochem. physiol., 37, 911, 1959)].

The original spot is colored when the chloroform methanol extract ofPSC-A (modified Bligh. Dyer method) is developed on a silica gel TLC byusing a mixture of petroleum ether, ether and ocetic acid (80:20:1)before being colored by treating with 50 % sulfuric acid solution andheating it.

(4) Description and Solubility

The agent is a pale yellow powder soluble in water, physiological salineand phosphate buffer, and is water soluble with a solubility of 1 mg/mlor higher. It is almost insoluble in methanol, ethanol, hexane andchloform.

(5) Coloring Reaction

Lowry, Folin reaction, ninhydrin reaction, phenol-sulfate reaction andanthron-sulfate reaction produce a positive result while Elson.Morganreaction yields a negative result.

(6) Isoelectric Point

pH 3.8˜4.2 (agarose electrofocusing)

(7) Stability

Stable in a neutral aqueous solution at room temperature for at least 24hours.

(8) Ultraviolet Spectrum

Maximum absorption occurs at about 272 nm as shown in FIG. 1.

(9) Enzymatic Activity

It does not cause the decompose of casein or collagen.

(10) Properties

(i) When 10 μg/ml of PSC-A is added to L cells of a cell-line derivedfrom mouse connective tissue before they are cultured for 24 hours of 2μg/ml of PSC-A is added to leukocytc cells derived from the spleen of anormal mouse before they are cultured for 24 hours, PSC-A does notexhibit any direct toxicity for either the L cells or the Leukocytecells.

(ii) For both a solution prepared by dissolving PSC-A in physiologicalsaline and a emulsion prepared by mixing PSC-A with Freund incompleteadjuvant or Freund complete adjuvant, and antibody which can react withP. aeruginosa appears in the serum of a mouse immunized with PSC-A.Additionally, PSC-A treated with the proteolytic cnzyme pronase orheated at 100° C. for 10 minutes does not react with a serum from amouse immunized with nontreated PSC-A.

(iii) The mouse monoclonal antibody having a specific affinity for PSC-Adoes not react with either the well-known common antigen OEP to P.aeruginosa or the endotoxin of P. aeruginosa (lipopolysaccharide).

When used as an agent for protecting against P. aeruginosa infection, itis preferable that this agent be administered by injection. A solutionor a lyophilized preparation prepared from PSC-A alone or combining itwith usual additive and excipient can be practically used. It ispossible to incorporate PSC-A in an oil-in-water type emulsion or awater in-oil type emulsion. Also, PSC-A can be practically used eitherby sealing PSC-A in liposome composed of phospholipid, cholesterol or byfixing PSC-A to the outer surface of the membrane of liposome.

The dose and the administration route for PSC-A may be properlyselected. It is preferable that the dose be 0.001˜10 mg per kg bodyweight. For the administration route, intracutaneous, subcutaneous,intravenous, intramuscular and intraperitoneal administrations can beperformed.

PSC-A has antigenicity which occurs in a so-called vaccine and inducesthe production of high concentration of an antibody for P. aeruginosa inthe serum of a mouse or guinea pig immunized with PSC-A.

Furthermore, PSC-A is highly active for protecting animal P. aeruginosainfection. For example, mice immunized with PSC-A were not infected witha lethal does of each of all serotypes of P. aeruginosa and could liveafter being treated with P. aeruginosa. This indicates remarkableability of PSC-A protect P. aeruginosa infection.

PSC-A has low acute toxicity because the 50% lethal does of PSC-A whenit is intravenously administered to mice is more than 5 mg/kg. As shownin claim, PSC-A does not cause any direct toxicity in animal cells.

From the above findings, it is considered that PSC-A is very useful asan agent for protecting against P. aeruginosa infection.

In the following, the method for manufacturing PSC-A will be describedaccording to examples and the utility of PSC-A as an agent forprotecting against P. aeruginosa infection will be described accordingto experimental examples.

EXAMPLE 1 Preparation of PSC-A from P. aeruginosa of Serotype E

The medium for the culture of P. aeruginosa was contained (per liter) 20g of sodium glutamate, 5 g of glycerin, 0.1 g of MgSO₄.7H₂ O, 0.55 g ofKH₂ PO₄, 5.6 g of Na₂ HPO₄.12H₂ O, 17.26 mg of Ca(NO₃)₂.4H₂ O and 50 μgof FeSO₄.7H₂ O, and was adjusted to pH 7.6.

After P. aeruginosa ATCC27581 (E type) was cultured on a nutrient agarmedium at 37° C. overnight, microbial cells were suspended inphysiological saline.

The, 0.5 ml of the suspension was inoculated into a Sakaguchi's flaskcontaining 150 ml of the synthetic medium having the above composition,and shaking culture was performed at 37° C. for 16 hours.

After the cultivation 1.2 ml of formalin was added to the culture of theflask, then the culture was thoroughly mixed before being left at roomtemperature for one hour. Then the culture was subjected tocentrifugation (12,000×g, 30 minutes) to collect microbial cells. Afterthat, by washing of the microbial cells with physiological saline anddistilled water, 55 g of wet microbial cells was obtained from 15 L ofculture medium. The wet microbial cells obtained thus were thensuspended in 220 ml of 20 mM Tris-Hc buffer (pH 8.0) containing 2% ofTriton X 100 and 10 mM of EDTA before being crushed with a DYNO-MILL(beads 0.1 mm φ) for three minutes while being cooled.

Next, the suspension of crushed microbial cells prepared thus was leftat 4° C. and the supernatant was collected by decantation. The residuewas suspended in 500 ml of above buffer and same procedure as above wasrepeated once. The supernatant collected thus was then pooled andcentrifuged (39,000×g, 30 minutes).

Next, 720 ml of the supernatant was applied to an affinity column packedwith affigel-10 (manufactured by Bio-Rad company) immobilized withmonoclonal antibody C-Ab(column size, 22×40 mm; 15 mg of C-Ab was boundto 1 ml of Affigel-10).

The column was then washed with 100 ml of 20 mM Tris-HCl buffer (pH 8.0)containing 0.5% Triton X100 and 100 ml of 20 mM Tris-HCl buffer (pH8.0), respectively and eluted with 60 mlof 50 mM glycine-HCl bufferedsaline (pH 3.0). The eluate obtained thus was then neutralized with 1Naqueous sodium hydrogen carbonate solution and dialyzed againstdistilled water at 4° C. for 24 hours. After that, the dialyzate waslyophilized to obtain 4.7 mg of a pure powder of PSC-A.

EXAMPLE 2 Preparation of PSC-A from P. aeruginosa of Serotype G

After P. aeruginosa ATCC 27584 (G type) was subjected to shaking culturein the synthetic medium (in the same way as in Example 1) at 37° C. for20 hours. The below procedure was performed in the same way as inExample 1. 6.2 mg of PSC-A was obtained from 58.2 g of wet microbialcells.

The amino acid composition of PSC-A was determined by an automatic aminoacid analyzer (IRICA A-5500) and the results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Amino acid composition of PSC-A                                               Amino acid          mole %*                                                   ______________________________________                                        Lysine              10.07                                                     Histidine           0.06                                                      Arginine            1.45                                                      Aspartic acid or asparagine                                                                       5.63                                                      Threonine           5.07                                                      Serine              3.59                                                      Glutamic acid or glutamine                                                                        15.44                                                     Proine              2.17                                                      Glycine             7.42                                                      Alanine             20.27                                                     Cystein             0.00                                                      Valine              11.57                                                     Methionine          1.45                                                      Isoleucine          4.18                                                      Leucine             7.10                                                      Tyrosine            0.46                                                      Phenylalanine       1.94                                                      Tryptophan**        2.12                                                      ______________________________________                                         *based on duplicate analysis of 24,48-, and 72h hydrolysate (in 6N HCl at     105° C.)                                                               **determined by a spectrophotometric method (Anal. Chem., 29, 1193, 1957)

EXAMPLE 3 Preparation of SPC-A from P. aeruginosa of Serotype I

After P. aeruginosa ATCC 27586 (I type) was subjected to shaking culturein the synthetic medium (in the same way as in Example 1) at 37° C. for20 hours. The below procedure was performed in the same way as inExample 1. 28 mg of PSC-A was obtained from 305 g of wet microbialcells.

EXAMPLE 4 Solution

After 1 mg of PSC-A obtained in Example 2 was dissolved in 10 ml ofphysiological saline, the solution prepared thus was subjected toaseptic filtration through a Nuclepore NO 20 (manufactured by Nucleporecompany). A 1 ml sample of the filtrate obtained thus was then pouredinto each vial without allowing any contamination by bacteria to obtaina solution of PSC-A.

EXAMPLE 5 Lyophilized preparation

After 10 mg of PSC-A obtained in Example 3 was dissolved in 10 ml ofdistilled water, 500 mg of mannitol was added to the solution.

The solution was filtered through a Nuclepore NO 20. A 1 ml sample ofthe filtrate obtained thus was then poured into each vial withoutallowing any contamination by bacteria before lyophilization wasperformed to obtain a lyophilized preparation of PSC-A.

EXAMPLE 6 Emulsion

After 1 mg of PSC-A obtained in Example 3 was dissolved in 0.5 ml ofphysiological saline, 0.5 ml of mixture solution consisting of liquidparaffin and Arlacel (manufactured by Arlacel A. Atlas ChemicalIndustries) in a ratio of 8.5 to 1.5 was added to the solution and awater-in-oil type emulsion was prepared.

(Effect of This Invention) EXPERIMENTAL EXAMPLE 2 Antigenic Property ofPSC-A in Mice

Equal amounts of Freund incomplete adjuvant and a physiological salinesolution of PSC-A obtained in Example 1 and that in Example 2 are mixedto prepare water-in-oil type emulsions, respectively.

After one group consisting of five BALB/lC female mice, 8 weeks old wasimmunized twice with each of the above emulsions at one-week intervals(10 μg per mouse of PSC-A was intraperitoneally administered for eachimmunization), blood was collected by eye bleeding (20 μl) from eachmouse four days following the final immunization. The titer of anti-P.aeruginosa antibody in the serum of the mouse was determined by DIBAmethod described in Experimental Example 1. The results are shown inTable 4. After the immunization with PSC-A was performed, a high titerof the antibody was detected in the serum. The antibody was not detectedin nonimmunized mice almost entirely.

                                      TABLE 4                                     __________________________________________________________________________                     Antibody Titer in Mouse Serum determined by                                   DIBA method*                                                 Immunogen  Mouse No.                                                                           IID 1002                                                                           ATCC 27581                                                                           ATCC 10145                                                                           ATCC 27586                                __________________________________________________________________________    PSC-A in Example 1                                                                       1     5.sup.3 × 10.sup.2                                                           5.sup.4 × 10.sup.2                                                             5.sup.4 × 10.sup.2                                                             5.sup.4 × 10.sup.2                             2     5.sup.3 × 10.sup.2                                                           5.sup.4 × 10.sup.2                                                             5.sup.3 × 10.sup.2                                                             5.sup.4 × 10.sup.2                             3     5.sup.3 × 10.sup.2                                                           5.sup.3 × 10.sup.2                                                             5.sup.3 × 10.sup.2                                                             5.sup.3 × 10.sup.2                             4     5.sup.3 × 10.sup.2                                                           5.sup.3 × 10.sup.2                                                             5.sup.3 × 10.sup.2                                                             5.sup.3 × 10.sup.2                             5     5.sup.3 × 10.sup.2                                                           5.sup.4 × 10.sup.2                                                             5.sup.4 × 10.sup.2                                                             5.sup.3 × 10.sup.2                  PSC-A in Example 2                                                                       6     5.sup.3 ×  10.sup.2                                                          5.sup.3 × 10.sup.2                                                             5.sup.4 × 10.sup.2                                                             5.sup.3 × 10.sup.2                             7     5.sup.3 × 10.sup.2                                                           5.sup.3 × 10.sup.2                                                             5.sup.3 × 10.sup.2                                                             5.sup.3 × 10.sup.2                             8     5.sup.3 × 10.sup.2                                                           5.sup.4 × 10.sup.2                                                             5.sup.4 × 10.sup.2                                                             5.sup.3 × 10.sup.2                             9     5.sup.3 × 10.sup.2                                                           5.sup.3 × 10.sup.2                                                             5.sup.3 × 10.sup.2                                                             5.sup.3 × 10.sup.2                             10    5.sup.3 × 10.sup.2                                                           5.sup.3 × 10.sup.2                                                             5.sup.3 × 10.sup.2                                                             5.sup.3 × 10.sup.2                  __________________________________________________________________________     *The antibody titer was determined by DIBA method using 0.3% formalin-        treated cells of P. aeruginosa IID1002 (B type), ATCC 27581 (E type), ATC     10145 (G type) and ATCC 27586 (I type).                                       The antibody titer for each bacterial stain was expressed as the maximum      multiple of dillution mouse serum for which dark blue spot was observed o     a nitrocellulose membrane filter.                                        

EXPERIMENTAL EXAMPLE 3 Effect of PSC-A on the protection against P.aeruginosa Infection (part 1)

After two groups each consisting of BALB/C female mice, 8 weeks old wereimmunized twice with each of the two emulsions shown in ExperimentalExample 2 at one-week intervals (10 μg per mouse of PSC-A wasintraperitoneally administered for each immunization), each mouse wasinfected with P. aeruginosa (5 LD₅₀) one week following the finalimmunization. Two strains of P. aeruginosa PA103 (E type) and P 28 (Ctype) were used for the infection. Each of these strains was cultured ona heart infusion agar medium (manufactured by Eiken Kagaku) overnightand collected and diluted with physiological saline. The 5 pts. of mucinwas added to 100 pts. of the bacterial suspension and each mouse wasintraperitoneally inoculated with about a 5 LD₅₀ amount of the bacterialsuspension prepared thus. For the control group, physiological salinealone was administered instead of PSC-A. The number of mice in eachgroup remaining days following infection by P. aeruginosa was countedand the results are shown in Table 5. Thus PSC-A was effective for theprotection against P. aeruginosa infection.

                  TABLE 5                                                         ______________________________________                                        Test              P. aeruginosa                                                                             Survival                                                                              Survival                                Groups Immunogen  Used        after 7 days                                                                          Rate(*)                                 ______________________________________                                        Control                                                                              --         PA103       0/5      0                                      I                 (E type)                                                    Group  PSC-A      PA103       5/5     100                                     I      in Example 1                                                                             (E type)                                                    Group  PSC-A      PA103       5/5     100                                     II     in Example 2                                                                             (E type)                                                    Control                                                                              --         P28(G type) 0/5      0                                      II     --                                                                     Group  PSC-A      "           5/5     100                                     III    in Example 1                                                           Group  PSC-A      "           5/5     100                                     IV     in Example 2                                                           ______________________________________                                    

EXPERIMENTAL EXAMPLE 4 Effect of PSC-A on the protection against P.aeruginosa Infection (part 2)

PSC-A obtained in Example 3 was dissolved in physiological saline toprepare a sample solution. After two groups each consisting of fiveBALB/C female mice, 8 weeks old were immunized four times with thesolution prepared thus at one-week intervals (20 μg per mouse of PSC-Awas subcutaneously administered for each immunization), these two groupswere infected with two strains of P. aeruginosa consisting of Pa103 (Etype) and P28 (G type), respectively. A bacterial suspension of each ofthese strains was prepared in the same manner as in Example 3 and eachmouse was infected with the bacterium by being intraperitoneallyinoculated with an about 3 LD₅₀ amount of the bacterial suspension. Forthe control group, physiological saline alone was administered insteadof PSC-A.

As seen in Table 6, PSC-A was effective for the protection against P.aeruginosa infection.

                  TABLE 6                                                         ______________________________________                                        Test              P. aeruginosa                                                                             Survival                                                                              Survival                                Groups Immunogen  Used        after 7 days                                                                          Rate(*)                                 ______________________________________                                        Control                                                                              --         PA103       0/5      0                                      I                 (E type)                                                    Group  PSC-A      PA103       4/5      80                                     I      in Example 3                                                                             (E type)                                                    Control                                                                              --         P28         0/5      0                                      II                (G type)                                                    Group  PSC-A      P28         5/5     100                                     II     in Example 3                                                                             (G type)                                                    ______________________________________                                    

EXPERIMENTAL EXAMPLE 5 Effect of PSC-A on the protection against P.aeruginosa infection (part 3)

Each of PSC-A obtained in Example 1 and that in Example 2 was dissolvedin physiological saline to prepare sample solutions. Two groups eachconsisting of 15 female mice, 6 weeks old were immunized three timeswith each of the two sample solutions at one-week intervals (30 μg permouse of PSC-A was intraperitoneally administered for eachimmunization). One week following the final immunization, each mouse wasinfected with P. aeruginosa. Two strains of P. aeruginosa PA103 (E type)and P28 (G type) were used for the infection similarly to ExperimentalExample 3 and 4. After suspension was prepared from these strains in thesame manner as in Experimental Example 3, a 3 LD₅₀ and 5.5 LD₅₀ amountof the suspension from the PA103 and P28 strain was interaperitoneallyinoculated into each mouse, respectively. For the control group,physiological saline alone was administered instead of PSC-A. The numberof mice in each group remaining 7 days following infection by P.aeruginosa was counted and results are shown in Table 7. Thus PSC-A waseffective for the protection against P. aeruginosa infection.

                  TABLE 7                                                         ______________________________________                                        Test              P. aeruginosa                                                                             Survival                                                                              Survival                                Groups Immunogen  Used        after 7 days                                                                          Rate(*)                                 ______________________________________                                        Control                                                                              --         PA103        0/15    0                                      I                 (E type)                                                    Group  PSC-A      PA103       15/15   100                                     I      in Example 1                                                                             (E type)                                                    Group  PSC-A      PA103       15/15   100                                     II     in Example 2                                                                             (E type)                                                    Control                                                                              --         P28          1/15   6.7                                     II                (G type)                                                    Group  PSC-A      P28         12/15   80                                      III    in Example 1                                                                             (G type)                                                    Group  PSC-A      P28         11/15   73.3                                    IV     in Example 2                                                                             (G tpe)                                                     ______________________________________                                    

EXPERIMENTAL EXAMPLE 6 Acute Toxicity

One group consisting of six BALB/C female mice, 5 weeks old wasintravenously administered with a solution prepared by dissolving PSC-Ain Example 3 in physiological saline. In the same way, LPS extractedfrom E. coli (lipopolysaccharide manufactured by Sigma company, serotypeNo. 0111 : B4) was intravenously administered to another mouse group.The LD₅₀ level of PSC-A and LPS for intravenous administration wasobserved during the 24 hours following the administration. The resultsare shown in Table 8. As distinct from LPS, PSC-A exhibited lowtoxicity.

                  TABLE 8                                                         ______________________________________                                        Sample       LD.sub.50 for Intravenous Administration                         ______________________________________                                        PSC-A        >5 mg/kg                                                         LPS from E. coli                                                                           ≦0.1 mg/kg                                                ______________________________________                                    

EXPERIMENTAL EXAMPLE 7 Pyrogenicity

One group consisting of three Japanese-while female rabbits wasintravenously administered with a solution prepared by dissolving PSC-Ain Example 3 in physiological saline. In the same way, LPS extractedfrom E. coli (lipopolysaccharide manufactured by Sigma company, serotypeNo. 0111 : B4) was intravenously administered to another rabbit group.The body temperature of each rabbit was successively measured from twohours before the administration until six hours after theadministration. The results are shown in Table 9. The pyrogenicity ofPSC-A was lower than that of LPS.

                  TABLE 9                                                         ______________________________________                                                   Minimum Dose for 0.6° C. increase of body                   Sample     temperature                                                        ______________________________________                                        PSC-A      >1 × 10.sup.2 μg/kg                                       LPS from E. coli                                                                         1 × 10.sup.-3 μg/kg                                       ______________________________________                                    

What is claimed is:
 1. PSC-A, a bacterial antigen obtained from P.aeruginosa, said antigen having the following properties:a. amino acidcomposition:

    ______________________________________                                        Amino acid composition of PSC-A                                               Amino acid          mole %                                                    ______________________________________                                        Lysine              10.07                                                     Histidine           0.06                                                      Arginine            1.45                                                      Aspartic acid or asparagine                                                                       5.63                                                      Threonine           5.07                                                      Serine              3.59                                                      Glutamic acid or glutamine                                                                        15.44                                                     Proline             2.17                                                      Glycine             7.42                                                      Alanine             20.27                                                     Cystein             0.00                                                      Valine              11.57                                                     Methionine          1.45                                                      Isoleucine          4.18                                                      Leucine             7.10                                                      Tyrosine            0.46                                                      Phenylalanine       1.94                                                      Tryptophan          2.12                                                      ______________________________________                                    

b. a molecular weight as determined by SDS-polyacrylamide gelelectrophoresis of about 15,000; c. a lipid content of 7.0 to 10.0%; d.a protein content of 55% as determined by colorimetric analysis byhydrolysis using ninhydrin; e. a sugar content of 3.0 to 5.0%; f. ahexosamine content of less than 1.0%; g. an isoelectric point ofpI=3.8-4.2 as determined by agarose electrofocusing; h. stability in aneutral aqueous solution at room temperature for at least 24 hours; i.maximum ultraviolet absorption at about 272 nm; j. no decomposition ofcasein or collagen; k. in appearance, a pale yellow powder soluble inwater, physiological saline, and phosphate buffer, water soluble with asolubility of at least 1 mg/ml; and almost insoluble in methanol, ether,hexane, or chloroform; l. producing a positive results in a Lowry-Folinreaction, ninhydrin reaction, phenol-sulfate reaction andanthron-sulfate reaction and a negative reaction in an Elson-Morganreaction; m. exhibiting no direct toxicity for either L cells of a cellline derived from mouse connective tissue before they are cultured for24 hours or for leukocyte cells derived from the spleen of a normalmouse before they are cultured for 24 hours; n. when said antigen istreated with pronase or heated at 100° C. for 10 minutes, there is notreaction with a serum from a mouse immunized with nontreated antigen; o.an antibody which can react with P. aeruginosa appears in the serum of amouse immunized with said antigen; p. wherein the mouse monoclonalantibody having a specific affinity for PSC-A does not react with eitherthe well-known common antigen OEP to P. aeruginosa or the endotoxin ofP. aeruginosa (lipopolysaccharide).
 2. A pharmaceutical compositioncontaining PSC-A as an active ingredient and a pharmaceuticallyacceptable carrier for protecting against P. aeruginosa infection. 3.The composition as set forth in claim 2 where the PSC-A is obtained frommicrobial cells of P. aeruginosa.